REPRODUCTIONRESEARCH Intracellular calcium and protein tyrosine phosphorylation during the release of bovine sperm adhering to the fallopian tube epithelium in vitro Roberto Gualtieri, Raffaele Boni1, Elisabetta Tosti2, Maria Zagami and Riccardo Talevi Dipartimento di Biologia Evolutiva e Comparata, Universita` di Napoli ‘Federico II’, Via Mezzocannone 8, 80134 Napoli, Italy, 1Dipartimento di Scienze delle Produzioni Animali, Campus Macchia Romana, 85100 Potenza, Italy and 2Stazione Zoologica ‘Anton Dohrn’, Villa Comunale, Napoli, Italy Correspondence should be addressed to R Gualtieri; Email: [email protected] Abstract In mammals, sperm adhesion to the epithelial cells lining the oviductal isthmus plays a key role in the maintenance of motility and in the selection of superior quality subpopulations. In the bovine species, heparin and other sulfated glycoconjugates powerfully induce the synchronous release of sperm adhering to tubal epithelium in vitro and may represent the signal which triggers release at ovulation in vivo. Sperm detachment may be due either to surface remodeling or to hyperactivation brought 21 21 about by capacitation. In this paper, the dynamics of intracellular free Ca concentration ([Ca ]i) and protein tyrosine phos- phorylation in sperm during and after heparin-induced release from in vitro cultured oviductal monolayers were assessed to determine whether this event is due to capacitation. Moreover, Ca21-ionophore A23187, thapsigargin, thimerosal and caffeine 21 were used to determine whether [Ca ]i increase and/or hyperactivation can induce sperm release. Results showed that: 1. 21 21 heparin-released sperm have significantly higher [Ca ]i than adhering sperm; 2. heparin induces a [Ca ]i elevation in the sperm head followed by detachment from the monolayers; 3. external Ca21 is not required for heparin-induced release; 4. 21 [Ca ]i increase and/or hyperactivation are unable to release sperm; and 5. heparin-released sperm have an increased level of tyrosine phosphorylated proteins compared with adhering sperm. In conclusion, although heparin is considered a long-lasting capacitation agent, it quickly modulates the capacitation of bovine sperm adhering to the fallopian epithelium, probably lead- ing to surface remodeling and therefore to the release of sperm selected and stored within the oviduct through adhesion. Reproduction (2005) 129 51–60 Introduction calcium content and reduced membrane protein tyrosine phosphorylation (pig: Petrunkina et al. 2001), superior The mammalian oviduct acts as a functional sperm reser- morphology (horse: Thomas et al. 1994), and normal chro- voir, providing a suitable environment that allows the matin structure (human: Ellington et al. 1998). Several maintenance of sperm fertilization competence until ovu- lines of evidence indicate that the ability of sperm to bind lation (Harper 1994). In several species, after mating, to and be released from oviductal epithelium is modulated sperm are sequestered in the lower oviduct through by capacitation. In fact, only uncapacitated sperm adhere adhesion to the epithelial cells lining its lumen (Wilmut & to oviductal cells (Smith & Yanagimachi 1991, Thomas Hunter 1984, Smith & Yanagimachi 1990). This event is et al. 1995, Lefebvre & Suarez 1996), whereas the detach- thought to prolong sperm life by delaying capacitation ment of sperm in the periovulatory period in vivo (Smith until unknown ovulation-associated signals induce the & Yanagimachi 1991), as well as the spontaneous release release of adhering sperm, allowing their progression during in vitro co-culture (Gualtieri & Talevi 2000), is toward the upper oviduct for fertilization (Smith & Yanagi- thought to be triggered by a remodeling of the sperm machi 1991, Harper 1994). Adhesion to the oviduct has plasma membrane and/or by hyperactivation brought been shown to play a key role also in the selection of pre- about by capacitation. In cattle, heparin-like glycosamino- existing sperm subpopulations characterized by intact glycans, normally present in the oviductal fluid, have acrosomes (cow: Gualtieri & Talevi 2000), an uncapaci- been regarded as potential in vivo capacitating agents tated status (horse: Thomas et al. 1995, cow: Lefebvre & (Parrish et al. 1988, 1989a). In a recent study, heparin, a Suarez 1996, pig: Fazeli et al. 1999), low internal free glycosaminoglycan routinely used to capacitate sperm in q 2005 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00374 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/23/2021 07:10:05PM via free access 52 R Gualtieri and others bovine in vitro fertilization (IVF), and other sulfated glyco- caffeine, thimerosal, Percoll, gelatin and Hoechst 33342 conjugates were shown to induce the release of sperm were obtained from Sigma; M199, fetal calf serum (FCS), adhering to the fallopian tube epithelium in vitro (Talevi & gentamycin, Fungizone, HEPES and sodium bicarbonate, Gualtieri 2001). This represented the first demonstration of from Gibco (Milan, Italy); Fluo 3 AM, from Molecular molecules able to cause the release of sperm adhering to Probe (Milan, Italy); thapsigargin, from Alomone Labs the oviductal epithelium among all species examined so (Milan, Italy); and antiphosphotyrosine monoclonal anti- far. Since heparin-like glycosaminoglycans are present in body (clone 1G2) and TRITC-conjugated goat antimouse the bovine oviductal fluid, and change in their concen- antibody, from Chemicon (Milan, Italy). Reagents and trations and capacitating activities is maximal at estrus water for preparation of salines and culture media were (Parrish et al. 1989a), sulfated glycosaminoglycans have all cell culture tested. been suggested to modulate progressively sperm capacita- tion, first inducing sperm release from the oviductal reser- Oviduct monolayers voir and then enhancing sperm fertilization ability (Talevi Oviducts were collected at the time of slaughter and trans- & Gualtieri 2001). Recently, the powerful sperm-releasing ported to the laboratory in Dulbecco’s PBS (D’PBS) sup- activity of sulfated glycosaminoglycans allowed us to plemented with 50 mg/ml gentamycin at 4 8C. Laminae of demonstrate that the sperm subpopulation selected by epithelial cells were recovered from oviducts of single ani- adhesion to in vitro cultured fallopian tube epithelium has mals by squeezing and cultured in M199 supplemented an enhanced zona pellucida binding and fertilization with 50 mg/ml gentamycin, 1 mg/ml Fungizone and 10% competence compared with the sperm subpopulation FCS, as previously described (Gualtieri & Talevi 2000). unable to adhere within the same ejaculate (Gualtieri & Bovine oviductal epithelial cells (BOEC) were cultured in Talevi 2003, Talevi & Gualtieri 2004). Therefore, sperm 10 cm Petri dishes (Falcon; Becton Dickinson Milan, Italy) adhesion and release can represent a crucial selective for 24–48 h and then transferred into four-well tissue cul- event during capacitation. Capacitation consists of a com- ture dishes (Nunclon, Roskilde, Denmark) with 12 mm, plex series of finely tuned events occurring during the gelatin-coated, German glass, round cover slips on the sperm transit through the female reproductive tract, and well bottom. Fresh media changes were performed every that are essential for sperm to develop the ability to bind 48 h. Cell confluence was attained in about 7–10 days. the zona pellucida, undergo the acrosome reaction and Monolayers were used within 24–48 h after attainment of fertilize the oocyte. Two well-recognized events accompa- cell confluence. Within each experiment, BOEC mono- nying capacitation of mammalian sperm are an increase layers from a single individual were washed three times in 2þ 2þ of sperm intracellular free Ca concentration ([Ca ]i) modified Tyrode’s albumin lactate pyruvate medium and the phosphorylation of protein tyrosine residues (Vis- (sperm-TALP: Parrish et al. 1988, modified as described in conti et al. 2002). The aim of the present paper was to test Paula-Lopes et al. 1998), and left in this medium until the hypothesis that heparin-induced release of sperm sperm addition (1–3 h). adhering to the fallopian tube epithelium in vitro rep- resents a very early event of capacitation. For this purpose, Sperm preparation experiments were designed to study the following: 1. the modification of sperm [Ca2þ] and protein tyrosine phos- Frozen bovine semen from three bulls (0.5 ml straws; i £ 6 phorylation levels during and/or after heparin-induced approximately 40 10 sperm per straw), obtained from sperm release; 2. the role played by extracellular Ca2þ in Semen Italy (San Giuliano Saliceta, Modena, Italy), was heparin-induced sperm release; and 3. the effect of the used in all experiments. Straws were thawed in a water pharmacologic agents Ca2þ-ionophore A23187, thapsigar- bath at 38 8C for 30 s and the semen was laid upon a dis- 2þ continuous (90/40) Percoll gradient with or without gin, thimerosal and caffeine that induce [Ca ]i increase and/or hyperactivation (Ho & Suarez 2001) on the release 10 mg/ml Hoechst 33342, and centrifuged for 30 min at of sperm adhering to the fallopian tube in vitro. The main 180 g. The supernatant was removed and the pellet, resus- findings demonstrate that heparin-induced release is pended in 2 ml BSA-free sperm-TALP, was centrifuged at 2þ 180 g for 10 min, and resuspended in 200 ml BSA-free accompanied by increase of both sperm [Ca ]i and tyro- sine phosphorylation, supporting